The highly conserved homologs of the secreted morphogen Hedgehog (Hh) are responsible for proper cellular differentiation during embryogenesis of both invertebrates and vertebrates (reviewed in Ingham and McMahon (2001) Genes Dev. 15:3059-3087). In mammals, three Hedgehog genes have been identified encoding Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) proteins. Their roles range from left-right asymmetry, neural tube patterning, limb patterning and branching morphogenesis to bone formation and spermatogenesis. Mutations of pathway components can lead to dramatic developmental disorders. While Hh signaling is mostly quiescent in adults, it has been implicated in many cancers, suggesting that inhibition of this pathway may have therapeutic benefit (reviewed in Beachy, P. A. et al. (2004) Nature 432:324-331; Pasca di Magliano and Hebrok (2003) Nat. Rev. Cancer 3:903-911; Rubin and de Sauvage (2006) Nat Rev Drug Discov. 5(12):1026-33).
Activation of the Hedgehog pathway through overexpression of Hedgehog is a hallmark of many cancers. The literature shows that overexpression of Hedgehog has been detected in many human tumor biopsies and cell lines of different types, including, for example, small cell lung cancer (SCLC), gastric and upper gastrointestinal tract cancer, pancreatic cancer, and prostate cancer.
The mature Hh amino terminal domain, responsible for proper short- and long-range signaling of Shh (sometimes referred to as Shh-N), is produced by autoproteolytic cleavage of the C-terminal intein-like domain (Lee, J. J. et al. (1994) Science 266:1528-1537; Porter, J. A. et al. (1995) Nature 374:363-366; Porter, J. A. et al. (1996) Cell 86:21-34) followed by addition of cholesterol at the C-terminus and palmitoylation at the N-terminus (Pepinsky et al. (1998) J. Biol. Chem. 273:14037-14045; Porter, J. A. et al. (1996) Science 274:255-259). The crystal structure of murine Shh revealed a tetrahedrally coordinated Zn2+ cation (Hall, T. M. et al. (1995) Nature 378:212-216) with an overall topology similar to those of the MD clan of metalloproteases (Rawlings, N. D. et al. (2008) Nucleic Acids Res. 36:D320-325), which include bacterial lysostahins (Bochtler, M. et al. (2004) Protein Sci. 13:854-861), and the VanX dipeptidase (Bussiere, D. E. et al. (1998) Mol. Cell. 2:75-84). Although Shh contains a metalloprotease pseudo-active site, mutations in the active site do not adversely affect biological assays signaling activity, suggesting that Shh acts as binding partner for membrane-bound receptors rather than as an enzymatically active protease (Fuse, N. et al. (1999) Proc. Natl. Acad. Sci. USA 96:10992-10999).
At the cell surface, the Hh signal is relayed by two multi-transmembrane prteins. The 12-pass transmembrane protein Patched (Ptch) is a negative regulator of the pathway, which in the absence of ligand prevents signaling by repressing the 7-passtranmembrane protein Smoothened (Smo) (Chen and Struhl (1996) Cell 87:553-563; Marigo, V. et al. (1996) Nature 384:176-179; Stone, D. M. et al. (1996) Nature 384:129-134). Binding of Shh to Ptch relieves the inhibition of Smo, allowing it to translocate to the primary cilium (Rohatgi and Scott (2007) Nat. Cell Biol. 9:1005-1009), where still poorly understood downstream signaling events ultimately lead to the activation of a family of Zn-finger transcription factors called Gli (Ingham and McMahon (2001) Genes Devel. 15: 3059-3087).
Regulation of the Hh signal at the cell surface by Ptch is finely tuned by a number of additional cell surface molecules such as the invertebrate proteins Ihog (interference Hedgehog) (Lum, L. et al. (2003) Science 299:2039-2045; Yao, S. et al. (2006) Cell 125:343-357) and Boi (brother of Ihog) (Yao, S. et al. (2006) Cell 125:343-357) and their corresponding vertebrate homologs Cdon (Kang, J. S. et al. (1997) J. Cell. Biol. 138:203-213; Zhang, W. et al. (2006) Dev. Cell 10: 657-65) and Boc (bioregional Cdon-binding protein) (Kang, J. S. et al. (2002) EMBO J. 21:114-124; Tenzen, T. et al. (2006) Dev. Cell 10:647-656). The co-receptors Ihog/Boi Cdon/Boc, and Gas1 (Growth arrest-specific-1 protein), which lacks a known homolog in Drosophila, are pathway agonists that enhance signal reception (Allen Allen, B. L. et al. (2007) Genes Dev. 21:1244-1257).
Like Ptch, Hedgehog-interacting protein (Hhip1, also known as Hip) is a negative regulator of the pathway and its transcription is upregulated in response to Hh signaling (Chuang, P. T. et al. (2003) Genes Dev. 17:342-347; Chuang, P. T. et al. (1999) Nature 397:617-621). Binding affinities for Shh to Hhip1 and Ptch on cells are similar, with KD values of 5 nM for Hhip1 and 4 nM for Ptch (Chuang and McMahon (1999) Nature 397:617-621). Decreased expression levels of Hhip1 have been noted in several human tumor tissue types, suggesting an important role for Hhip1 in suppressing tumor development (Olsen, C. L. et al. (2004) BMC Cancer 4:43).
Hhip1 is a 700 residue protein with an N-terminal signal peptide and a large extracellular region that ends with a hydrophobic C-terminal stretch (Chuang and McMahon (1999) Nature 397:617-621). In addition to the predominantly membrane-associated form, a soluble form of Hhip1 has also been detected in mature brains of adult rodents (Coulombe, J. et al. (2004) Mol. Cell. Neurosci. 25:323-333). Other than the likely presence of two epidermal growth factor (EGF) domains and four potential N-linked glycosylation sites (Chuang and McMahon (1999) Nature 397:617-621), nothing is known about the structural arrangement of the Hhip1 extracellular domain (ECD).
Although the structure of Shh has been known for some time, a precise understanding of the molecular interactions with its membrane-associated receptors has remained elusive. Recently, a comparison of structures of fibronectin type III (FNIII) domain 1 of Ihog and FNIII domain 3 of Cdon in complex with Drosphila Hh and vertebrate Shh, respectively, have revealed completely different binding modes (McLellan, J. S. et al. (2006) Proc. Natl. Acad. Sci. USA 103:17208-17213).
Until now, the crystal structure of Hhip1 has remained unsolved and it was not known how Hhip1 interacted with Hh to exert its effect. Likewise, Ptch has not been crystallized, so it was also unknown how Ptch interacted with Hh.
Thus, in order to effectively combat Hedgehog pathway-associated tumors, there is an urgent need in the art to better understand the precise interaction of Hh with Hh-controlling molecules such as Hhip1 and Ptch. A more complete understanding of the structural/functional relationship of the members of the Hedgehog signaling pathway, and more specifically, a better understanding of the molecular interaction between Ptch and Hhip1 with Hh would provide the necessary information for the rational design of small molecule and large molecule inhibitors of Hh and therapies for Hh-related cancers and conditions.